Evaluation of humoral immune status in porcine epidemic diarrhea virus (PEDV) infected sows under. field conditions

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1 Evlution of humorl immune sttus in porine epidemi dirrhe virus (PEDV) infeted sows under field onditions Kng Ouyng, Dun-Ling Shyu, Sntosh Dhkl, Jgdish Hiremth, Bsvrj Binjwdgi, Yshvnth S. Lkshmnpp, Rui Guo, Russell Rnsurgh, Kthryn M. Bondr, Phillip Guger, et l. To ite this version: Kng Ouyng, Dun-Ling Shyu, Sntosh Dhkl, Jgdish Hiremth, Bsvrj Binjwdgi, et l.. Evlution of humorl immune sttus in porine epidemi dirrhe virus (PEDV) infeted sows under field onditions. Veterinry Reserh, BioMed Centrl, 15, (1), pp.1. <1.11/s x>. <hl-1319> HAL Id: hl Sumitted on Jul 1 HAL is multi-disiplinry open ess rhive for the deposit nd dissemintion of sientifi reserh douments, whether they re pulished or not. The douments my ome from tehing nd reserh institutions in Frne or rod, or from puli or privte reserh enters. L rhive ouverte pluridisiplinire HAL, est destinée u dépôt et à l diffusion de douments sientifiques de niveu reherhe, puliés ou non, émnnt des étlissements d enseignement et de reherhe frnçis ou étrngers, des lortoires pulis ou privés.

2 DOI 1.11/s x Veterinry Reserh SHORT REPORT Open Aess Evlution of humorl immune sttus in porine epidemi dirrhe virus (PEDV) infeted sows under field onditions Kng Ouyng 1,, Dun Ling Shyu 1, Sntosh Dhkl 1, Jgdish Hiremth 1, Bsvrj Binjwdgi 1, Yshvnth S. Lkshmnpp 1, Rui Guo 3, Russell Rnsurgh 3, Kthryn M. Bondr 1, Phillip Guger, Jinqing Zhng, Terry Speht 5, Aron Gilertie 5, Willim Minton 5, Ying Fng 3 nd Gourpur J. Renukrdhy 1* Astrt Porine epidemi dirrhe virus (PEDV) is n eonomilly devstting enteri disese in the swine industry. The virus infets pigs of ll ges, ut it use severe linil disese in neontl sukling pigs with up to 1% mortlity. Currently, ville vines re not ompletely effetive nd feedk methods utilizing PEDV infeted mteril hs vrile suess in preventing reinfetion. Comprehensive informtion on the levels nd durtion of effetor/memory IgA nd IgG ntiody sereting B ell response in the intestines nd lymphoid orgns of PEDV-infeted sows, nd their ssoition with speifi ntiody levels in linil smples suh s plsm, orl fluid, nd fees is importnt. Therefore, our gol in this study ws to quntify PEDV speifi IgA nd IgG B ell responses in sows t pproximtely 1 nd months post-infetion in ommeril swine herds, inluding prity one nd higher sows. Our dt indited tht evlution of oth PEDV speifi IgA nd IgG ntiody levels in the plsm nd orl fluid (ut not fees) smples is enefiil in disese dignosis. PEDV speifi B ell response in the intestine nd spleen of infeted sows deline y months, nd this ssoites with speifi ntiody levels in the plsm nd orl fluid smples; ut the virus neutrliztion titers in plsm remins high eyond months post-infetion. In onlusion, in sows infeted with PEDV the presene of effetor/memory B ell response nd strong virus neutrliztion titers in plsm up to months postinfetion, suggests their potentil to protet sows from reinfetion nd provide mternl immunity to neontes, ut hllenge studies re required to onfirm suh responses. Introdution Porine epidemi dirrhe (PED) linilly mnifests s severe wtery dirrhe with susequent dehydrtion in ll ges of swine, ut highly severe in suking pigs [1]. Other linil signs of PED inlude vomiting nd norexi. PED is hrterized y the mortlity rte of 3 1% in neontes, nd high moridity ut low mortlity in wened pigs []. Eonomi losses due to elevted mortlity nd deresed prodution y PEDV re signifint in the US *Correspondene: gourpur.1@osu.edu 1 Food Animl Helth Reserh Progrm (FAHRP), OARDC, Deprtment of Veterinry Preventive Mediine, The Ohio Stte University, Wooster, OH 91, USA Full list of uthor informtion is ville t the end of the rtile swine herds. PED virus (PEDV) is the ustive gent of PED. PEDV ws deteted on multiple US swine frms in April of 13 [1, 3], nd the virus hs ontinued to spred through swine produing sttes t n lrming rte until the end of 1. Over PEDV tests hve een onduted in the US etween My 13 nd Mrh 1, nd reported 757 ses (~1%) positive in 7 sttes []. PEDV is n enveloped virus hving k genome nd enode four struturl proteins, spike (S), envelope (E), memrne (M), nd nuleopsid (N) [5, ]. The S protein of PEDV is the priniple surfe glyoprotein involved in virus tthment nd entry, nd it ontins virus-neutrlizing B ell epitopes [7 9]. 15 Ouyng et l. This rtile is distriuted under the terms of the Cretive Commons Attriution. Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pulidomin/zero/1./) pplies to the dt mde ville in this rtile, unless otherwise stted.

3 Pge of 11 PEDV ontinues to infet nive swine frms rehing strit ioseurity protools for unknown resons or hs re-infeted reeding frms fter implementing feedk strtegies. Piglets re expeted to e proteted from the linil disese through olostrl immunity reeived from immune dms [1]. However, protetion from infetion nd shedding hs een vrile with osionl filure of feedk regimens. Control nd prevention of PEDV is one of the mjor hurdles to the swine industry in the US. Currently, ville vines re not ompletely effetive nd feedk methods utilizing PEDV infeted mteril hs shown vried suess in preventing reinfetion. This ould e ttriuted to non-vilility of relile dignosti tools to monitor the protetive herd immune sttus in sows. Moreover, informtion out levels nd durtion of PEDV herd immune sttus in sows is importnt to implite pproprite ontrol mesures t verge of disese outreks. Therefore, it is ritil to develop stndrdized isotype ntiody trgeted ssys to determine the ssoition of linil smples dt with PEDV speifi B ell response t the intestines nd lymphoid tissues of sows reovered from PED under field onditions. In this study we quntified oth PEDV speifi IgA nd IgG ntiody levels in the linil smples (plsm, orl fluid, nd fees) nd ssoited tht to the isotype speifi B ell responses in the intestine nd lymphoid tissues of PED infeted sows in ommeril reeding herds of two different prities (primiprous nd multiprous). Mterils nd methods Cells Vero ells (ATCC CCL-1) were ultured in Minimum Essentil Medi (Gio, CA, USA) supplemented with 1% het intivted fetl ovine serum (Atlnt Biologils, GA, USA), mm l-glutmine (Gio) nd ntiioti/ntimyoti solution (HyClone, UT) t 37 C in humidified tmosphere with 5% CO. For preprtion of virus stoks nd in virus neutrlizing (VN) ssy, the MEM ws supplemented with toylsulfonyl phenyllnyl hloromethyl ketone (TPCK)-trypsin (1 μg/ml) (Sigm, MO, USA),.3% tryptose phosphte roth (Sigm), % yest extrt (BD, MD) nd ntiioti/ntimyoti solution (HyClone, UT, USA). Virus PEDV strin KS 1-1 ws isolted from PED infeted field fel smples t Knss Stte University, nd ws propgted in Vero ells. Confluent ell monolyer ws wshed with sterile phosphte-uffered sline (PBS) twie efore infeting with the virus, nd fter 1 h of dsorption t 37 C dditionl infetion medium ws dded without removing the inoulum. The virus indued ytopthogeni effet ws rehed to pproximtely 9% in 3 dys. The virus ulture superntnt ws olleted fter freeze-thwing two times, nd then lrified t high speed entrifugtion (3 g for 3 min) nd purified the virl ntigen y ultrentrifugtion through % (wt/vol) surose ushion (1 g for h). Both the stok virus nd virl ntigen were stored t C until used. Virl protein onentrtion ws mesured y miro BCA protein ssy kit (Thermo Sientifi, IL, USA) nd virus titer ws determined y IFA s desried previously [11 13]. Animls Three swine reeding frms loted in the Middle Estern prt of the Unites Sttes were hosen for this study, with the two frms hving the history of piglet mortlity nd lortory onfirmtion of PEDV infetion y ELISA t pproximtely 1 nd months efore neropsy, nd the third frm hd no history of PED infetion. Six sows eh from primiprous nd multiprous group with totl of 3 sows with the history of PEDV infetion or no infetion were used in this study (Tle 1). Animls were euthnized in slughter plnt (Bo Evns slughterhouse, Xeni, Ohio) ording to the stndrd proedures with neessry efforts to minimize suffering of nimls. All the proedures on nimls used in this study were pproved y the Committee on the Ethis of Animl Experiments of The Ohio Stte University. On the dy of neropsy, plsm, orl sw, nd fel smples were olleted for virus speifi ntiody isotype titrtion; nd ileum, mesenteri lymph nodes (MLN), nd spleen smples were olleted in MEM ontining ntiiotis nd ntifungl for isolting mononuler ells (MNCs). Isoltion of immune ells MNCs from ileum, MLN, nd spleen were isolted s previously desried [1 17] with few modifitions. Briefly, ileum, spleen, nd MLN tissues were ut into tiny piees nd only ileum tissues were treted with Type II ollgense fter treting with EDTA nd dithiothreitol. Cell suspensions were otined fter pssing the digested tissues of ileum, spleen, nd MLN through stinless steel Celletors fitted with n μm mesh sreen (Celletor, FL, USA). The hrvested MNCs were sujeted to density grdient entrifugtion with 3% nd 7% Peroll, nd the ells in the interfe were olleted nd filtered through μm ell striner (BD Flon, MA, USA) nd re-suspended in enrihed-rpmi (E-RPMI, RPMI ontining 1% FBS, μm HEPES, 1 mm sodium pyruvte, 5 μm -ME, 1x non-essentil mino id, nd 1x ntiioti nd ntifungl). The viility of ells ws onfirmed y trypn lue dye exlusion method, nd ounted using hemoytometer.

4 Pge 3 of 11 Tle 1 Grouping of sows t different stges of PEDV infetion. Groups No. of sows PEDV infetion Sttus Arevition 1 Mok Primiprous Mok-PP Mok Multiprous Mok-MP 3 1 month postinfetion Primiprous 1 m PI-PP 1 month postinfetion Multiprous 1 m PI-MP 5 months postinfetion Primiprous m PI-PP months postinfetion Multiprous m PI-MP A totl of 3 sows from three different swine reeding frms loted in the Mid-Estern prt of the US, hving ler PEDV infetion history (uninfeted, 1 month or months post-infeted) were seleted, nd six sows eh from primiprous nd multiprous groups were trnsported to slughter plnt in Ohio. Clinil smples suh s lood (plsm), orl fluid, nd fees, nd tissues of ileum, mesenteri lymph nodes, nd spleen were olleted on the dy of neropsy. In vitro stimultion of MNCs with PEDV ntigen MNCs isolted from ileum, MLN, nd spleen were plted in -well ell ulture plte (5 1 ells/well) in ml E-RPMI in the presene of semi-purified PEDV virl ntigen (5 μg/ml), nd ells treted with medium lone or lipopolyshride (5 μg/ml) were inluded s ontrols. Cells were ultured for dys t 39 C with 5% CO, nd.5 ml of E-RPMI ws dded to eh well on every seond dy. Superntnts were olleted to mesure PEDV speifi IgA nd IgG ntiody y ELISA. Cells were hrvested, wshed using PBS, re-suspended in E-RPMI, ounted nd used for deteting the popultion of PEDVspeifi IgA nd IgG ntiody sereting ells (ASC) y enzyme linked immunospot (ELISPOT) ssy, nd for eluidting the frequeny of IgA + nd IgG + B ells y flow ytometry. Antiody isotype ELISA PEDV speifi IgA nd IgG ntiody levels were determined s desried previously [15, 1]. Briefly, pretitrted mounts of PEDV whole virus-derived ntigen (5 μg/ml), reominnt PEDV S protein (5 μg/ml) or M protein (1 μg/ml) diluted in 5 mm ronte uffer (ph 9.) were oted overnight t C in the 9-well ELISA pltes (Corning, MA, USA). Pltes were loked using 1% nonft milk in PBS ontining 5% Tween. Plsm smple ws diluted 1:, 1:, 1:3, nd 1:1, nd orl fluid nd fel smples were diluted 1:, 1:3, 1:1 nd 1:51. The superntnts hrvested from stimulted MNCs were diluted 1: nd 5 μl of eh of the smples ws pplied into duplite wells nd inuted for 1 h t room temperture (RT). Pltes were wshed nd horserdish peroxidse-onjugted got nti-pig IgA (Bethyl lortories) or IgG ntiody (KPL) (1:5) ws dded to the pltes nd inuted for 1 h. The retion ws developed using TMB peroxidse sustrte nd stopped using 1 M phosphori id nd pltes were red t OD 5. Virus titrtion nd virus neutrlizing (VN) ntiody ssys PEDV titer in stoks nd VN ntiody titer in linil smples were nlyzed y IFA s desried previously [11 13] with few modifitions. Briefly, onfluent Vero ell monolyers in 9-well pltes were wshed one using PBS efore seeding the smples. For virus titrtion, tenfold serilly diluted virus stok ws dded to ells plte in qudruplite; for VN titrtion, linil smples were UV treted (5 nm for 5 min) nd het intivted (5 C for 3 min), nd twofold serilly diluted smples were inuted with equl volume of PEDV (5 TCID 5 per well) for 1.5 h t 37 C, nd the mixture ws trnsferred into Vero ells grown in mirotiter pltes in duplite. Pltes were inuted for 1 h t 37 C nd infetion medium ws dded fter the wells were wshed one using PBS nd inuted for h t 37 C. Pltes were wshed nd fixed using etone-milli-q wter (:) mixture for 1 min t RT, nd dried ompletely efore immunostined. Cells were treted with nti-pedv N protein speifi monolonl ntiody (SD-9) (Medgene l, SD, USA) (1:1) for h t 37 C, followed y tretment with Alex Fluor onjugted got nti-mouse IgG (H + L) (Invitrogen, CA, USA) seondry ntiody (1:). The plte ws exmined under fluoresent mirosope fter mounting with glyerol-pbs (:). The virus indued ytopthi effet ws exmined under fluoresent mirosope, nd the titer ws lulted using the Reed nd Muenh method. The virl titer ws expressed in 5% tissue ulture infetive dose (TCID 5 ) per ml. The VN titer ws determined to e the reiprol dilution of plsm/orl fluid/fel smples tht indued greter thn 9% inhiition of virl infetion s shown in virus ontrol wells (Figure ). Enzyme linked immunospot (ELISPOT) ssy for quntifying PEDV speifi ASCs ELISPOT ssy ws performed to nlyze the popultion of PEDV speifi IgA nd IgG ntiody sereting B ells s desried previously [1 17] with few modifitions. Briefly, two kinds of PEDV whole virus-derived ntigen oted pltes were used in this study. (i) Vero ells infeted nd fixed ell ulture pltes: PEDV (1 μl of 1 TCID 5 /ml) ontining 1 μg/ml of TPCK-trypsin ws inoulted to onfluent Vero ell monolyers in 9-well tissue ulture plte. Cells were fixed with % etone t 1 h post-infetion nd pltes were stored

5 Pge of 11 t C. At tht stge out % of ells were infeted s tested y IFA, nd the ell monolyer ws still intt. Mok-infeted ells treted extly the sme wy were used s ontrol. (ii) PEDV whole virus-derived ntigen immunoptured pltes: Semi-purified virl ntigen (5 μg/ml) diluted in 5 mm ronte uffer (ph 9.) ws oted overnight t C in nitroellulose-sed 9-well mirotiter pltes (Millipore, MA, USA). Pltes were wshed with PBS nd loked with E-RPMI for 1 h t RT nd stimulted MNCs of ileum, MLN, nd spleen were plted t three tenfold dilutions in duplite wells strting from ells/well. All pltes were inuted for 1 h t 39 C with 5% CO, nd wshed with PBS ontining 5% Tween (PBST). The ntiody prodution ws deteted using horserdish peroxidseleled ffinity-purified got nti-pig IgA (Bethyl lortories, Texs) or IgG (KPL, Mrylnd) diluted 1: in PBST nd inuted for h t 37 C. The olor ws developed using 3-mino-9-ethylrzole sustrte (Sigm-Aldrih, St. Louis, USA) nd spots were ounted using the AID ELISPOT Reder System (Autoimun Dignostik GmH Strsserg, Germny). Dt were expressed s the men numers of ASCs per MNCs. Flow ytometri nlyses The frequenies of IgA + nd IgG + B ells from 1 quired events of immunostined MNCs were determined y flow ytometry s desried previously [1] with few modifitions. Briefly, MNCs were stimulted with PEDV whole virus-derived ntigen s desried ove nd immunostined with mouse nti-pig IgA ma (Clone K 1F1, AD Serote) followed y got ntimouse IgG1 onjugted to APC/CY7 nd rit nti-pig IgG onjugted to Texs Red. Susequently, ells were fixed, permeilized nd then intrellulr stined using FITC onjugted rt nti-mouse CD79β ntiody (Clone AT17-, AD Serote), whih ws shown to ross-ret with pig B ells [1]. Cells were quired using BD Ari II flow ytometer nd nlyzed using the FlowJo softwre. Ethis sttement This study ws rried out in strit ordne with the reommendtions y Puli Helth Servie Poliy, United Sttes Deprtment of Agriulture Regultions, the Ntionl Reserh Counil s Guide for the Cre nd Use of Lortory Animls, nd the Federtion of Animl Siene Soieties Guide for the Cre nd Use of Agriulturl Animls in Agriulturl Reserh nd Tehing nd ll the relevnt institutionl, stte, nd federl regultions nd poliies regrding re nd use of nimls t the Ohio Stte. Sttistil nlysis All dt were expressed s the men ± stndrd error of men (SEM) of six sows. Sttistil nlyses were performed y one-wy ANOVA followed y Tukey s post ho test (GrphPd InStt 5. prism softwre), nd the P vlue of <5 ws onsidered signifint. Results Antiody response in PEDV infeted sows Detils of sow groups used in this study re provided in Tle 1. Clinilly, sows were helthy efore trnsporttion to the slughter plnt t Xeni in Ohio nd rested for dy efore neropsy. PEDV speifi IgA nd IgG ntiody responses were determined in plsm, orl fluid, nd fel smples using PEDV ntigen, reominnt PEDV S nd M protein oted pltes. Our results indited PEDV speifi IgA ntiody response in the smples of plsm (Figure 1A) nd orl fluid (Figure 1B) from oth one nd months post-infeted sows ws signifintly higher thn uninfeted sows, nd similr trend ws present in reominnt S (ut not M) protein oted pltes (Figures 1D nd E). Surprisingly, in fel smples the speifi IgA ntiody ginst PEDV whole virusderived ntigen (Figure 1C), S protein (Figure 1D), nd M protein (Figure 1E) ws sent. Although PEDV speifi IgG ntiody levels in the plsm of infeted sows ws signifintly higher thn uninfeted sows (Figures 1F, I nd J), the kground optil density vlues of IgG from uninfeted sows ws higher ompred to respetive IgA levels (Figures 1A, D nd E). In orl fluid smples of months post PEDVinfeted primiprous sows, speifi IgG ntiody levels ginst whole virl ntigen nd reominnt S nd M proteins were signifintly higher thn uninfeted nd other three infeted sow groups (Figures 1G, I nd J). PEDV speifi IgG ntiody in fel smples ws sent in infeted sows (Figures 1H, I nd J). Our dt suggests tht for dignosti ntiody nlysis of PEDV infetion in sows, the plsm nd orl fluid smples (ut not fel smples) re relile nd onsistent. Further, VN ntiody titers ginst PEDV in infeted sow smples were quntified y IFA. For this ssy, 5 TCID 5 of PEDV in eh well ws used, whih showed sustntil mount of infeted ells t h post-infetion (Figure ). Our dt reveled tht in plsm of PEDV uninfeted sows, the kground VN titer ws up to 1. But, in infeted sows, the VN titer ws very high in ll four infeted groups with titer of 51 (Figure 3A), suggesting tht even fter months the VN titer in oth primiprous nd multiprous sows remined high in the plsm. The VN titers in orl fluid nd fel smples were < in ll the infeted sows (Figures 3B nd C).

6 Pge 5 of 11 PEDV speifi IgA A B C D E OD F G H I J 1.5 **.5. * * OD Dilution: 1: 1: 1:3 1:1 1: 1:3 1:1 1:51 1: 1:3 1:1 1:51 1: 1: 1: 1: 1: 1: Coting: PEDV ntigen PEDV ntigen PEDV ntigen PEDV S protein PEDV M protein Smple: PEDV speifi IgG Plsm Orl sw Fel sw Plsm Orl sw Fel sw Plsm 1. Mok-PP. Mok-MP 3. 1m PI-PP. 1m PI-MP 5. m PI-PP. m PI-MP Figure 1 Quntifition of PEDV-speifi IgA nd IgG ntiody titers in linil smples of PEDV infeted sows. Smples were olleted from the uninfeted nd post PEDV-infeted sows s indited in the Tle 1. PEDV speifi IgA (A E) nd IgG (F J) ntiody titers were quntified y ELISA, with pltes oted either using PEDV whole virus-derived ntigen (A C nd F H), S protein (D, I) or M protein (E, J); in plsm (A, D F, I, J), orl fluid (B, D, E, G, I, J), nd fel (C E nd H J) smples. Eh r represents men OD 5 vlue ± SEM from six sows. Lowerse lphet indites sttistilly signifint differene etween uninfeted nd PEDV-infeted orresponding prity sow groups, nd sterisk indites sttistil differene etween the indited PEDV-infeted sow groups ( or *P < 5, or **P < 1 nd or P < 1). * Orl sw Fel sw Flow ytometry nlysis of PEDV speifi IgA nd IgG positive B ells in infeted sows Flow ytometry nlysis llows simultneous multi-prmetri nlysis of the physil nd hemil hrteristis of immune ells. MNCs isolted from ileum, MLN, nd spleen were stimulted ex vivo using PEDV whole virus-derived ntigen for dys, nd susequently nlyzed the frequeny of effetor/memory IgA + nd IgG + B ells y flow ytometry. Ileum MNCs of PEDV infeted sows stimulted with the virus ntigen hd signifintly higher frequenies of oth CD79 + IgA + nd CD79 + IgG + B ells ompred to uninfeted sows (Figures B nd H). In ddition, the frequeny of CD79 + IgG + B ells (ut not CD79 + IgA + B ells) were lso signifintly higher in ileum MNCs unstimulted with medium ontrol of three groups of PEDV infeted sows (exept 1 month multiprous sows) ompred to uninfeted sows (Figures A nd G). Speifilly, ntigen speifi CD79 + IgA + nd CD79 + IgG + B ell popultions in the ileum of 1 month post PEDV-infeted multiprous sows were signifintly higher thn months post-infeted nimls with oth primiprous nd multiprous sttus (Figures B nd H). However, there ws no signifint hnges in the frequeny of oth CD79 + IgA + nd CD79 + IgG + B ells in the MLN MNCs of PEDV infeted sows, either stimulted with virl ntigen (Figures D nd J) or unstimulted medium ontrol (Figures C nd I). In the spleen of ll four post-pedv-infeted sow groups the frequeny of CD79 + IgA + B ells ws signifintly higher ompred to their uninfeted ge-mthed ounterprts, either unstimulted or stimulted with PEDV whole virus-derived ntigen (Figures E nd F). However, in oth primiprous nd multiprous sows t 1 month post PEDV-infetion, signifintly higher frequeny of CD79 + IgG + B ells ws oserved ompred to oth uninfeted nd months post-infeted ounterprts, either unstimulted or stimulted with PEDV whole virus-derived ntigen (Figures K nd L). Further, in the spleen of multiprous sows t 1 month post PEDV-infetion, inresed frequeny of CD79 + IgA + B ells in unstimulted MNCs ws signifintly higher thn tht of primiprous nd multiprous sows t months post-infetion (Figure E). In spleen of primiprous sows t months post PEDV-infetion, ntigen speifi CD79 + IgA + memory B ell popultion in ntigen-stimulted splenoytes ws signifintly less ompred to tht in multiprous sows t 1 nd month post-infetion (Figure F). Overll, our dt indited tht in PEDVinfeted multiprous sows, CD79 + IgA + B ell response is stronger thn tht of primiprous infeted ounterprt.

7 Pge of 11 A TCID 5 Virus ontrol Phse ontrst IFA B Smple dilution PEDV VNT negtive PEDV VNT positive titer of 51 Phse ontrst IFA Phse ontrst IFA No virus 1 Figure Representtive pitures showing virus neutrlizing ntiody response in PEDV infeted sows plsm smples. A Fifty TCID 5 of virus ws used in the NA ssy whih showed ppreile quntity of infeted ells y immunofluoresene (IFA) ssy t h post-infetion. B Eh of representtives experimentl plsm smple of PEDV uninfeted nd infeted sows were twofold serilly diluted from 1: to 1:1 nd mixed with 5 TCID 5 of PEDV; nd Vero ells treted with tht mixture were sujeted to IFA nd exmined under mgnifition. VN titer ws expressed s the reiprol of the highest dilution rtio of test smples tht used greter thn 9% redution in virus indued fluoresene foi units ompred to tht of virus ontrol. VN titers A Plsm B Orl sw Fel sw C Mok-PP. Mok-MP 3. 1m PI-PP. 1m PI-MP 5. m PI-PP. m PI-MP Figure 3 Virus neutrlizing ntiody titers in PEDV infeted sows. (A) Plsm, (B) Orl fluid nd (C) fel smples were nlyzed for the VN titers ginst PEDV y immunofluoresene ssy. The VN titer ws determined s the reiprol dilution of the test smple tht indued greter thn 9% inhiition of virl infetion. Eh r represents men VN titers ± SEM from six sows. Lower se lphet indites sttistilly signifint differene ( P < 5 nd P < 1) etween mok-uninfeted nd PEDV-infeted orresponding primiprous nd multiprous sow groups. In the ulture superntnts hrvested t dy six ultures of ileum, MLN, nd spleen MNCs from multiprous sows t 1 month post-infetion, signifintly inresed seretion of PEDV speifi IgA ntiodies speifi to the virus ws deteted (Figure 5A). While in primiprous PEDV infeted sows splenoytes stimulted with ntigen, signifintly inresed IgA ntiody seretion ws deteted (Figure 5B). While numerilly inresed (ut not signifint) trends in IgA ntiody prodution ws oserved in PEDV infeted sows derived MNCs of ll

8 Pge 7 of 11 % of lymphoytes % of lymphoytes 1. Mok-PP. Mok-MP 3. 1m PI-PP. 1m PI-MP 5. m PI-PP. m PI-MP PEDV speifi IgA + B ells y Flow ytometry A B C D E F PEDV speifi IgG + B ells y Flow ytometry G H I J K L * MNCs: Ileum Ileum MLN MLN Spleen Spleen Stimulte: Medium ontrol PEDV ntigen Medium ontrol PEDV ntigen Medium ontrol PEDV ntigen Figure Flow ytometry nlyses to determine the frequeny of IgA + nd IgG + B ells in muosl tissues of PEDV infeted sows. Mononuler ells were isolted from ileum (A, B, G, H), mesenteri lymph nodes (C, D, I, J), nd spleen (E, F, K, L), nd stimulted ex vivo with PEDV whole virus-derived ntigen (B, D, F, H, J, L) or medium ontrol (A, C, E, G, I, K) for dys. The frequeny of B ells positive for IgA (A F) nd IgG (G L) ntiodies were determined y flow ytometry. Eh r represents the men of perent of lymphoytes positive for IgA or IgG positive B ells ± SEM from six sows. Lowerse lphet indites sttistilly signifint differene etween uninfeted nd PEDV-infeted orresponding prity sow groups, nd sterisk indites sttistil differene etween the indited PEDV-infeted sow groups ( or *P < 5, or **P < 1 nd or P < 1) ** * ** three tissues ginst the S protein, nd ginst M protein the ntiody levels were very low (Figures 5C nd D). In the splenoytes from primiprous sows t 1 month post PEDV-infetion, signifintly higher seretion of virus speifi IgG ntiodies ws deteted (Figure 5F). Quntifition of PEDV sereting IgA nd IgG ASC in infeted sows ELISPOT ssy llows visuliztion of the seretory produt(s) of individul tivted ells, nd thus provides oth qulittive nd quntittive informtion on T nd B ell responses. Popultion of PEDV speifi IgA nd IgG sereting ells (ASCs) present in every.5 million MNCs of ileum, MLN nd spleen were enumerted y ELISPOT ssy (Figure ). To perform this ssy, we used oth PEDV whole virus-derived ntigen ontining fixed Vero ells plte nd semi-purified PEDV ntigen immunoptured plte. Both the methods deteted similr trend in the frequenies of oth IgA nd IgG ASCs in PEDV infeted sow groups, ut in fixed Vero ell ntigen oted pltes, fivefold to tenfold higher numers of ASCs were deteted ompred to the other, therefore fixed ell ntigen plte dt is shown here (Figure ). As expeted we did not detet PEDV speifi IgA nd IgG ASCs in oth uninfeted primiprous nd multiprous sows (Figure ). Overll, the popultion of PEDV speifi IgA ASCs ws greter thn IgG ASCs in ll three tissue derived MNCs (Figure ). In post 1 month PEDV-infeted multiprous sows ileum MNCs, ultured for dys without ny further ntigeni stimultion, the numer of virus-speifi IgA ASCs (~) were signifintly greter ompred to ontrol uninfeted nd months post PEDV-infeted (1 ASCs) sows (Figure A); nd similr trend ws present in stimulted ileum MNCs (Figure B). In ontrst, the numer of virus-speifi IgG ASCs were too low (<5) in the ileum MNCs (Figures G nd H). In MLN MNCs, the numer of PEDV-speifi IgA ASCs in multiprous sows t 1 nd months post PEDV-infetion were signifintly higher ompred to uninfeted ontrol sows (Figures C nd D). In unstimulted MNCs of spleen, frequeny of PEDV-speifi IgA ASCs (~) in ll four PEDVinfeted sow groups were signifintly higher thn tht from uninfeted ontrol sows (Figure E); nd only in PEDV-infeted multiprous sows, signifint inrese in IgA ASCs in virl ntigen-stimulted MNCs ws oserved ompred to uninfeted nimls (Figure F). However, signifintly greter numers of PEDV-speifi IgG ASCs (~3) were present only in the PEDV whole virus-derived ntigen stimulted MNCs from multiprous sows t 1 month post PEDV-infetion in omprison to tht of uninfeted ontrol sows (Figure L).

9 Pge of 11 OD5 A 1. * ** 1. Mok-PP. Mok-MP 3. 1m PI-PP. 1m PI-MP 5. m PI-PP. m PI-MP B 1. PEDV speifi IgA C * D.. * * * E F PEDV speifi IgG G.15 H.15 OD Coting: PEDV ntigen PEDV ntigen PEDV S protein PEDV M protein MNCs: Ileum MLN Spleen Ileum MLN Spleen Ileum MLN Spleen Ileum MLN Spleen Stimulte: Control unstimulted PEDV ntigen PEDV ntigen PEDV ntigen Figure 5 Anlysis of PEDV speifi IgA nd IgG ntiodies sereted y stimulted MNCs of ileum, mesenteri lymph nodes, nd spleen. Culture superntnts were hrvested on dy six from MNCs stimulted ex vivo with PEDV whole virus-derived ntigen (B D, F H) or ontrol unstimulted (A, E). PEDV speifi IgA (A D) nd IgG (E H) levels were determined y ELISA, with pltes oted either with PEDV whole virus-derived ntigen (A, B nd E, F), S protein (C, G) or M protein (D, H). Eh r represents men OD 5 vlue ± SEM from six sows. Lowerse lphet indites sttistilly signifint differene etween uninfeted nd PEDV-infeted orresponding prity sow groups, nd sterisk indites sttistil differene etween the indited PEDV-infeted sow groups ( or *P < 5, or **P < 1 nd or P < 1). Disussion We evluted PEDV speifi B ell response in infeted sows t pproximte 1 nd months post-infetion, omprising of oth primiprous nd multiprous sow groups nd inluded 1 ge-mthed ontrol uninfeted sows. In n erlier study, PEDV whole virus ntigen sed ELISA ws developed using the virus grown in Vero ells, nd the virl ntigen used in the ssy ws extrted using.% Triton X-1 to nlyze ntiodies in 1 field smples t the herd level [19]. Though we followed similr method of growing the virus, ut the ELISA ntigen ws extrted from freeze-thwed virl ulture fluid sujeted to % surose ushion ultrentrifugtion, proly the ell deris still present in our ntigen preprtion would hve ontriuted to the oserved high kground tivity in IgG ELISA in plsm, whih needs further investigtion. PEDV S protein is lss I virl fusion protein [], nd is leved y host-derived proteses when virus enters the suseptile ell [1]. The S protein ontins B ell epitopes for indution of neutrlizing ntiodies []. The S protein-sed ELISA ws found to e sensitive to evlute ntiody response ginst PEDV in plsm nd olostrum smples [3 ]. In our study, in ddition to whole virus ntigen, oth reominnt PEDV S nd M protein sed IgA nd IgG ELISA ws used to estimte the levels of ntiody in plsm, orl fluid, nd fel smples of sows; nd deteted high levels of speifi IgA response in infeted sows plsm followed y in orl fluid, ut not in fel smples. This suggests tht PEDV speifi ntiodies in the fel smples of infeted sows dispper erly (pproximtely 1 months) post-infetion; in spite of the presene PEDV speifi IgA nd IgG ASCs in intestines nd lymphoid orgns t months post-infetion. We lso notied tht PEDV speifi IgA response in plsm (ut not in orl fluid) remined high until months. Furthermore, though we found omprle retivity of PEDV whole virus derived ntigen nd S protein for detetion of speifi IgA ntiodies, the ntiody levels were twofold less in the S protein sed ELISA (ODs t 1: dilution). Overll, our dt suggested tht for dignosis purpose it is idel to use oth whole virus nd S protein sed IgA nd IgG ELISA in herd orl fluid s well s in sttistilly eptle numer of plsm smples.

10 Pge 9 of 11 ASCs / 5x1 5 MNCs ASCs / 5x1 5 MNCs A 3 1 G * PEDV speifi IgA sereting ells y ELISPOT 1. Mok-PP B. Mok-MP C D E F m PI-PP. 1m PI-MP 5. m PI-PP. m PI-MP PEDV speifi IgG sereting ells y ELISPOT H I J K L MNCs: Ileum Ileum MLN MLN Spleen Spleen Stimulte: Medium ontrol PEDV ntigen Medium ontrol PEDV ntigen Medium ontrol PEDV ntigen Figure ELISPOT nlyses to quntify PEDV speifi IgA nd IgG ntiody sereting ell popultion in ileum, mesenteri lymph nodes, nd spleen of PEDV infeted sows. MNCs isolted from ileum (A, B, G, H), MLN (C, D, I, J), nd spleen (E, F, K, L) were stimulted ex vivo with PEDV whole virus-derived ntigen (B, D, F, H, J, L) or medium ontrol (A, C, E, G, I, K) for dys. PEDV speifi IgA (A F) nd IgG (G L) ntiody sereting ells (ASCs) were deteted y ELISPOT. Eh r represents men numer of PEDV-speifi ASCs per MNCs from six sows. Lowerse lphet indites sttistilly signifint differene etween uninfeted nd PEDV-infeted orresponding prity sow groups, nd sterisk indites sttistil differene etween the indited PEDV-infeted sow groups ( or *P < 5, or **P < 1 nd or P < 1) Consistent with the results of others [1, 7, ], we did not detet PEDV speifi response in ileum MNCs of uninfeted sows, onfirming their PEDV negtive sttus nd suggesting the need of in vivo priming of the immune system y the live virus to oserve the response. But like erlier results in PEDV infeted experimentl piglets [1], we oserved signifintly inresed popultion of PEDV speifi ASCs nd IgA nd IgG positive B ells in the ileum nd spleen of sows in in vitro ultured MNCs for dys in the sene of virl ntigen restimultion, inditing the presene of virus speifi effetor B ells for up to months in PEDV infeted sows. In one of our previous study in pigs vinted/infeted with porine reprodutive nd respirtory syndrome virus similr rell lymphoyte response in lung MNCs nd PBMC ultured in vitro in the sene of the virus ntigen ws oserved [1, 9]. Interestingly, we deteted sustntilly higher frequeny of PEDV speifi ASCs ompred to the results of n erlier study [1], this ould e ttriuted to differene in the ge of pigs nd the virus strin used in the study. At this stge due to lk of pig speifi B ell memory mrker regent we ould not nlyze the frequeny of memory B ell pool y flow ytometry in PEDV infeted sows. When PEDV speifi IgA nd IgG responses in linil smples were ompred with the ntiody sereting B ell popultion in the ileum, MLN, nd spleen of sows nlyzed y oth flow ytometry nd ELISPOT ssys; surprisingly, multiprous sows hd inresed humorl response oth in terms of ntiody nd B ell response ompred to primiprous nimls, nd tht response ws high in 1 month post-infeted sows nd delined y months. But the VN titers in plsm remined high in ll the PEDV-infeted sows, suggesting tht infeted sows ould e resistnt to reinfetion eyond months. However, the level of protetion vries depending on extent of virl ntigeni diversity, helth sttus of sows, nd seondry miroil infetions. Due to lk of virus isoltion from PEDV-infeted sows, the geneti vriility etween the virus tht infeted sows nd the virus used for ex vivo stimultion ould not e nlyzed, nd thus we ould not determine the levels of ross-protetion to PEDV in sows. In n erlier experimentl study, 11 dys old onventionl pigs were infeted with PEDV, nd speifi IgA nd IgG ASCs in the intestines were deteted t higher levels thn in systemi sites (spleen nd PBMC) [1]. However, in sows nturlly infeted with PEDV, we oserved higher levels of IgA nd IgG ASCs in spleen thn in intestines, suggesting tht systemi response is eqully importnt nd it persists for longer time in infeted sows ompred to piglets. Sukling nd gnotoioti pigs infeted

11 Pge 1 of 11 with TGEV nd rotvirus, respetively, hd higher numers of speifi IgG ASCs ompred to IgA ASCs in the intestines [1, 1, 7, 3]. But in our study, in oth one nd months post PEDV-infeted primiprous nd multiprous sows, the frequeny of speifi IgA ASCs were greter thn IgG ASCs in the intestines, MLN, nd spleen. This suggests the enhned suseptiility of neontl pigs to PEDV is responsile for indution of greter levels of IgG ASCs in the intestinl tissues in omprison to tht of sows whih experiene low to mild level of PEDV-infetion. Sine PEDV infetion is lolized to intestinl muos, stimultion of systemi immune response espeilly in piglets is t very low levels [31, 3]. However, our study in sows suggested tht systemi response in spleen is predominnt over the response in intestines; the differenes might e due to time frme of smpling nd the ge of the nimls. Immunologilly, the possile reson ould e the migrtion of tivted PEDV ntigen-ering dendriti ells to spleen, resulting in tivtion of nïve B nd T ells. Alterntively, the memory lymphoytes from the muos- ssoited lymphoid tissues ould migrte to spleen nd one mrrow [33]. In summry, PEDV speifi IgA nd IgG B ells in infeted sows were detetle t higher levels in ileum nd spleen ompred to tht in MLN using oth ELISPOT (totl B ell response) nd flow ytometry (detetion of frequeny of speifi IgA nd IgG B ell response) ssys, with the lter ssy demonstrting more roust result ompred to the former in deteting ntigen nd isotype speifi B ells. However, detetion of ntiody isotype speifi B ell response with memory phenotype mrker in muosl tissues will e highly enefiil to determine PEDV vine or infetion indued memory in pigs. In onlusion, our results suggests tht detetion of PEDV speifi IgA in plsm nd orl fluid smples hs potentil dignosti implition, nd estimtion of VN titers in plsm ould serve to determine the protetive immune sttus. Although PEDV speifi B ell response deline y months in oth ileum nd spleen of nturlly infeted sows, the presene of high levels of VN titers suggest tht suh sows my protet their offspring from PEDV infetion y genetilly losely relted virus y up to months. Competing interests The uthors delre tht they hve no ompeting interests. Author detils 1 Food Animl Helth Reserh Progrm (FAHRP), OARDC, Deprtment of Veterinry Preventive Mediine, The Ohio Stte University, Wooster, OH 91, USA. College of Animl Siene nd Tehnology, Gungxi University, Nnning, Chin. 3 Deprtment of Dignosti Mediine nd Pthoiology, College of Veterinry Mediine, Knss Stte University, Mnhttn, KS 5, USA. Veterinry Dignosti nd Prodution Animl Mediine, Iow Stte University, Ames, IA, USA. 5 Four Str Veterinry Servies, Chiksw, OH 5, USA. Authors ontriutions KO performed the experiments, nlysis of dt (inluding sttistil nlysis) nd drfting of the mnusript. DLS, SD, JH, BB, YSL, nd KMB performed the experiments. RG, RR, PG, JZ, nd YF prepred nd provided the regents exlusively for this reserh. TS, AG, nd WM helped in identifying the infeted sow herd performed smple olletion. GJR performed the experiments, nlysis of dt, edited nd finlized the mnusript. All uthors red nd pproved the finl mnusript. Aknowledgements This work ws supported y grnt wrd from Ntionl Pork Bord (NPB Projet#1-17). Slries nd reserh support were lso provided y stte nd federl funds pproprited to Ohio Agriulturl Reserh nd Development enter, The Ohio Stte University. Reeived: 5 August 15 Aepted: 17 Novemer 15 Referenes 1. 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